Page 1
ii
WHEY PROTEIN FRACTIONATION BASED ON SP-SEPHAROSE CATION
EXCHANGE CHROMATOGRAPHY
HAZMAN BIN ABD MULOK
A thesis submitted in fulfillment
of the requirements for the award of the Degree of
Bachelor of Chemical Engineering
Faculty of Chemical & Natural Resources Engineering
Universiti Malaysia Pahang
NOVEMBER 2010
Page 2
v
ABSTRACT
Bovine whey protein consists of different type of proteins such as β-
lactoglobulin (β-lag), α-lactalbumin (α-lac), immunoglobulin, bovine serum albumin
(BSA), lactoferrin, lactoperoxidase and glycomacropeptide. Single protein is more
valuable compare to the mixture of proteins and it can be used for specific application.
The main objective of this study is to fractionate whey protein components into several
groups or single protein fraction using SP-sepharoseTM
Fast Flow (Amersham
Biosciences) cation exchange chromatography column. The chromatographic process
was run at different pH range from pH 4 to pH 8 using 24 ml column. All major whey
proteins were recovered in the elution fraction at pH 4 and pH 4.5. No protein was
bound at pH 7 and pH 8. The best whey fractionation from 2 ml whey feed was
achieved at pH 4 with the yield for β-lag, BSA and α-lac are 83.33%, 66.44% and
10.51% respectively. Different protein fraction recovered during fractionation process at
different pH using cation exchange chromatography process, can be used as a guideline
to isolate particular protein of interest from bovine whey.
Page 3
vi
ABSTRAK
Whey protein daripada susu lembu terdiri daripada beberapa jenis protein seperti
β-lactoglobulin (β-lag), α-lactalbumin (α-lac), immunoglobulin, bovine serum albumin
(BSA), lactoferrin, lactoperoxidase dan glycomacropeptide. Protein tunggal adalah lebih
bernilai jika dibandingkan dengan campuran protein dan ia boleh digunakan untuk
aplikasi tertentu. Objektif utama kajian ini dijalankan adalah untuk mengasingkan
komponen whey protein kepada beberapa kumpulan atau protein tunggal dengan
menggunakan SP-sepharoseTM
Fast Flow (Amersham Biosciences) cation exchange
chromatography column. Proses kromatografi dijalankan pada pH berbeza antara pH 4
hingga pH 8 menggunakan kolum bersaiz 24 ml. Kesemua protein utama whey di
perolehi di dalam pecahan elusi pada pH 4 dan pH 4.5. Tiada protein yang terikat pada
kromatografi kolum pada pH 7 dan pH 8. Pengasingan terbaik dari 2 ml whey di capai
pada pH 4 dimana peratusan yield masing-masing untuk β-lag, BSA dan α-lac adalah
88.33%, 66.44% dan 10.51%. Pecahan protein yang berlainan komposisi yang di
perolehi dari proses pengasingan pada pH yang berlainan menggunakan proses cation
exchange kromatografi boleh di gunakan sebagai panduan untuk mendapatkan protein
yang tertentu daripada whey protein.
Page 4
vii
TABLE OF CONTENTS
PAGE
DECLARATION ii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES ix
LIST OF FIGURES xi
LIST OF ABBREVATIONS xii
LIST OF APPENDICES xiv
CHAPTER 1 INTRODUCTION 1
1.1 Research Background 1
1.2 Problem Statements 2
1.3 Objective of Study 2
1.4 Scopes of Study 2
CHAPTER 2 LITERATURE REVIEW 3
2.1 Composition of Bovine Milk 3
2.1.1 β-Lactoglobulin
2.1.2 α-Lactalbumin
2.1.3 Bovine Serum Albumin
2.1.4 Immunoglobulins
2.1.5 Other Proteins
3
5
5
5
5
Page 5
viii
2.2 Application of Bovine Whey Protein 6
2.3 Protein Separation Techniques 6
2.3.1 Chromatography
2.3.2 Membrane Separation
2.3.3 Other Techniques
8
9
9
2.4 Ion Exchange Chromatography for Protein Fractionation 10
2.5 Mechanism of Ion Exchange Chromatography 11
2.5.1 Protein Elution Strategies 13
2.6 Advantages of Ion Exchange Chromatography 14
2.7 Preparation of Buffers
2.7.1 Theory of Buffering
2.7.2 Buffer Selection
14
15
16
CHAPTER 3 METHODOLOGY 19
3.1 Preparation of Bovine Whey Protein 19
3.2 Preparation of Buffer Solutions 19
3.3 Cation-Exchange Chromatography 21
3.4 Reverse Phase Chromatography (RPC) 21
CHAPTER 4 RESULTS AND DISCUSSIONS 23
4.1
4.2
4.3
4.4
4.5
4.6
Protein Fractionation at pH 4
Protein Fractionation at pH 4.5
Protein Fractionation at pH 5
Protein Fractionation at pH 5.5
Protein Fractionation at pH 6
Protein Fractionation at pH 7 and pH 8
23
25
27
30
33
35
CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 38
5.1 Conclusions 38
5.2 Recommendations 39
REFERENCES 40
APPENDICES 42
A Results and Calculations from RPC Analysis 42
B Standard Curve 49
Page 6
ix
LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Typical concentration of proteins in bovine milk 4
2.2 Protein composition of bovine whey 4
2.3 Several techniques for protein purification 7
2.4 Conditions under which a protein binds or does not bind
to the ion exchange matrix
12
2.5 Some common buffers with wide range of buffering
capacity
15
2.6 Limitations of buffers commonly used in extraction 16
3.1 pH range and type of buffers used 20
3.2 pH table and recipes for preparation of acetate buffers 20
3.3 pH table and recipes for preparation of phosphate buffers 20
4.1 Summary of protein fractionation for pH 4 24
4.2 Summary of protein fractionation for pH 4.5 26
4.3 Summary of protein fractionation for pH 5 29
4.4 Summary of protein fractionation for pH 5.5 32
4.5 Summary of protein fractionation for pH 6 34
Page 7
x
4.6 Summary of protein fractionation for pH 7 36
4.7 Summary of protein fractionation for pH 8 36
4.8 Percentage yield of collected β-lag and BSA 37
5.1 Summary of experimental results 41
Page 8
xi
LIST OF FIGURES
FIGURE NO. TITLE PAGE
2.1 Guideline for choosing a cation or anion exchange
column based on the pI of the target protein
11
2.2 Schematic diagram illustrating mechanism of ion
exchange
13
3.1 SP-sepharose fast flow column 21
4.1 Chromatogram from SP-sepharose cation exchange
chromatography run for whey protein at pH 4
23
4.2 Chromatogram from SP-sepharose cation exchange
chromatography run for whey protein at pH 4.5
27
4.3 Chromatogram from SP-sepharose cation exchange
chromatography run for whey protein at pH 5
28
4.4 Chromatogram from SP-sepharose cation exchange
chromatography run for whey protein at pH 5.5
30
4.5 Chromatogram from SP-sepharose cation exchange
chromatography run for whey protein at pH 6
33
4.6 Chromatogram from SP-sepharose cation exchange
chromatography run for whey protein at pH 7
35
4.7 Chromatogram from SP-sepharose cation exchange
chromatography run for whey protein at pH 8
36
Page 9
xii
LIST OF ABBREVIATIONS
α-lac - α-lactalbumin
β-lag - β-lactoglobulin
Ag+ - Silver
BSA - Bovine Serum Albumin
CH3COO- - Acetate
Cl- - Chloride
CO2 - Carbon dioxide
DEAE - Diethylaminoethyl cellulose
F- - Fluoride
H+ - Hydrogen
HCl - Hydrochloric acid
HCOO- - Formate ion
HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HPLC - High Performance Liquid Chromatography
I- - Iodine
Ig - Immunoglobulins
IgA - Immunoglobulin A
IgG - Immunoglobulin G
IgM - Immunoglobulin M
K+ - Potassium
KH2PO4 - Potassium Phosphate, mono-sodium salt
K2HPO4 - Potassium Phosphate, di-sodium salt
L - Litre
Li+ - Lithium
M - Molarity
MeCN - Acetonitrile
MES - 2-(N-morpholino)ethanesulfonic acid
ml - Millilitre
MOPS - 3-(N-morpholino)propanesulfonic acid
Na+ - Sodium
NaCl - Sodium Chloride
NaH2PO4 - Sodium Phosphate, mono-sodium salt
NaOH - Sodium Hydroxide
Na2HPO4 - Sodium Phosphate, di-sodium salt
NH4+ - Ammonium
NO3- - Nitrate
OH- - Hydroxide
pI - Isoelectric Point
PIPES - Piperazine-N,N′-bis(2-ethanesulfonic acid)
Page 10
xiii
PO43-
- Phosphate
RPC - Reverse Phase Chromatography
SDS-PAGE - Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
SP - Sulphopropyl
TFA - Trifluoroacetic acid
Tris - Tris(hydroxymethyl)aminomethane
UV - Ultraviolet
Page 11
xiv
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Results and Calculations from RPC Analysis 42
B Standard Curves 49
Page 12
1
CHAPTER 1
INTRODUCTION
1.1 Research Background
Caseins and whey proteins are two major proteins present in the bovine milk. In
whey proteins, different types of proteins exist such as β-lactoglobulin (β-lag), α-
lactalbumin (α-lac), immunoglobulins (Ig), bovine serum albumin (BSA), lactoferrin,
lactoperoxidase, glycomacropeptide. These whey proteins have very high
pharmaceutical value (Hahn et al., 1996). However, the separation process involves in
fractionation them into individual protein is very challenging.
There are several techniques or methods commonly used to separate whey
proteins component such as by using chromatography or membrane based separation
and also precipitation techniques. Chromatography technique, particularly ion-
exchange adsorption process, has been developed and utilized successfully on a
commercial scale for whey proteins separation and whey protein concentrates
production (Gerberding and Byers, 1998).
Based on the fact that single proteins component can give more benefit compare
to its present in the mixture form, this study was conducted to fractionate the whey
protein component by using SP sepharose cation exchange chromatography.
1.2 Problem Statements
Single protein isolation from bovine whey protein is very challenging task.
Some components of whey protein have very similar properties and exist at various
Page 13
2
levels of concentrations. By exploiting the different in protein isoelectric point (pI), SP
sepharose cation exchange chromatography was used in this study to fractionate whey
protein into several fraction or single protein component. This defined fraction or pure
protein fraction can further be used for specific application and will have a better value
compared to its original composition.
1.3 Objective of Study
The main objective of this study is to fractionate whey protein components into
several groups of protein or single protein fraction using SP sepharose cation exchange
chromatography by optimizing the operation pH during the purification process.
1.4 Scopes of Study
In order to fulfill the research objective, the following scopes were outlined:
1. Preparation of whey protein solution from bovine milk.
2. Setup and operated cation exchanger chromatography using AKTA Explorer
100 liquid chromatography system.
3. Study the fractionation of bovine whey protein at different pH from pH 4 to
pH 8.
4. Analysis of protein fraction by using reverse phase chromatography
techniques.
Page 14
3
CHAPTER 2
LITERATURE REVIEW
2.1 Composition of Bovine Milk
Bovine milk consists of water, proteins, carbohydrates, lipids, vitamins, minerals
and growth factors. Bovine milk generally contains 30-35 g L-1
protein and is
commonly divided into two classes based on the solubility at pH 4.6 which is the
insoluble caseins that represent approximately 80% of total milk protein, and the soluble
whey proteins that represent approximately 20% of total milk protein (Robinson, 2002,
Walstra et al., 2006). Both the casein and whey protein groups are heterogeneous
(Robinson, 2002). Bovine whey proteins consist of different types of proteins such as β-
lactoglobulin (β-lag), α-lactalbumin (α-lac), immunoglobulins (Ig), bovine serum
albumin (BSA), lactoferrin, lactoperoxidase, and glycomacropeptide. Table 2.1 shows
the concentrations of different proteins in milk and Table 2.2 shows the protein
composition and properties of bovine whey according to Hahn et al. (1998).
2.1.1 β-Lactoglobulin
β-Lag is the most abundant whey protein and represents about 50% of the total
whey protein in bovine milk. There are eight known genetic variants of β-Lag: A, B, C,
D, E, F, G and Dr. The A and B genetic variants are the most common and exist at
almost the same frequency. β-Lag has a molecular weight of 18 kDa and contains two
internal disulfide bonds and a single free thiol group, which is of great importance for
changes occurring in milk during heating (Robinson, 2002).
Page 15
4
Table 2.1: Typical concentration of proteins in bovine milk (Robinson, 2002)
Grams/Liter % of total protein
Total protein 33 100
Total caseins 26 79.5
αs1-Casein 10 30.6
αs2-Casein 2.6 8.0
β-Casein 9.3 28.4
κ-Casein 3.3 10.1
Total whey proteins 6.3 19.3
α-Lactalbumin 1.2 3.7
β-Lactoglobulin 3.2 9.8
BSA 0.4 1.2
Immunoglobulins 0.7 2.1
Proteose peptone 0.8 2.4
Table 2.2: Protein composition of bovine whey according to Hahn et al. (1998).
Protein Average
concentration in
whey (g/L)
Molecular
mass (×10-3
)
Isoelectric
point,
pI
α-lactalbumin (α-lac) 1.5 14.2 4.7-5.1
Bovine Serum Albumin (BSA) 0.3-0.6 69 4.9
β-lactoglobulin (β-lag) 3-4 18.4 5.2
Immunoglobulins (Ig) 0.6-0.9 150-900 5.8-7.3
Lactoferrin 0.05 78 8.0
Lactoperoxidase 0.06 78 9.6
Page 16
5
2.1.2 α-Lactalbumin
α-Lac accounts for about 20% of the whey proteins and has three known genetic
variants. It has a molecular weight of 14 kDa and contains four interchain disulfide
bonds. α-Lac binds two atoms of calcium very strongly, and it is rendered susceptible to
denaturation when these atoms are removed (Robinson, 2002).
2.1.3 Bovine Serum Albumin
BSA represents about 5% of the total whey proteins and is identical to the serum
albumin found in the blood. The protein is synthesized in the liver and gains entrance to
milk through the secretory cells. It has one free thiol and disulfide linkages, which hold
the protein in multiloop structure (Robinson, 2002). Serum albumin appears to function
as a carrier of small molecules, such as fatty acids, but any specific role that it may play
is unknown (Robinson, 2002).
2.1.4 Immunoglobulins
Igs which are approximately 10% of the whey protein are antibodies synthesized
in response to stimulation by macromolecular antigens foreign to the animal. They also
are polymers of two kinds of polypeptide chains which is light (L) of molecular weight
22.4 kDa and heavy (H) of molecular weight 50-60 kDa. Four types of Ig have been
found in bovine milk are IgM, IgA, IgE, and IgG (Robinson, 2002).
2.1.5 Other Proteins
Several other proteins such as β-microglobulin, lactoperoxidase, lactoferrin, and
transferring, both of which are iron-binding proteins, proteose peptones, and a group of
acyl glycoproteins are found in small quantities in whey (Robinson, 2002).
Page 17
6
2.2 Application of Bovine Whey Protein
Single proteins extracted from whey protein can give benefits to human and also
animal nutrition. Besides that, it has a high value in pharmaceutical industries. Oral
administration of bovine IgG is known to be an effective treatment of various infections
for new-born infants whereas lactoferrin and lactoperoxidase are known to act as
antimicrobial factors (Hahn et al., 1998).
2.3 Protein Separation Techniques
In protein separation, a variety of technique can be use to isolate a single protein
of interest from a complex mixture. Protein separation is vital for the characterization
of the function, interactions and structure of the interested protein. Protein separation is
typically the most laborious aspect in bioproduct manufacturing. By exploiting the
difference in size, physico-chemical properties and binding affinity of particular protein,
precise protein separation can be achieved (Ahmed, 2005). Two most common
technique used for protein separation is chromatography and membrane filtration.
Table 2.3 shows several techniques for protein purification.
Page 18
7
Table 2.3: Several techniques for protein purification (Ahmed, 2005)
Technique Property
required
Remarks Recommended
application
Membrane
filtration
Molecular size Fractionation as
well as
concentration.
Loss of protein by
non-specific
adsorption.
At the beginning of a
purification procedure.
Particularly useful for
concentrating large
volumes of culture
medium.
Centrifugation Molecular size,
shape, density
Commonly used
for cellular
fractionation.
Preparative
isoelectric focusing
pI Proteins precipitate
in the rotofor
chamber
Size exclusion
chromatography
Molecular size Usually low
resolution. Provide
information about
protein molecular
weight.
At the end of a
purification procedure
Ion exchange
chromatography
Charge Protein binding
capacity usually
high
At the beginning of a
purification procedure
Reversed phase
chromatography
Hydrophobicity Resolution varies
according to gel
size. Commonly
used for peptide
separation.
Used for separation of
peptides, digested
purified proteins, and
other applications
where loss of protein’s
biological activity is
not a concern.
Hydrophobic
interaction
chromatography
Hydrophobicity After ammonium
sulfate fractionation,
but before ion-
exchange
chromatography
Affinity
chromatography
Binding ligand Usually specific
separation.
Limited by
availability of
immobilized
ligand. Expensive
to scale up.
At the beginning of a
purification procedure
Chromatofocusing Charge, pI Useful to separate
isoforms of closely
spaced pIs. Use after
affinity
chromatography
Page 19
8
2.3.1 Chromatography
Chromatography is operated based on the partitioning of a sample between a
moving phase and a stationary phase. Nowadays, chromatography is recognized as the
most powerful separation method with regard to resolution and versatility, having
superior resolving power and capable of isolating larger quantities of protein. Different
types of chromatographic techniques have evolved, including paper, thin layer, and
liquid chromatography (Rosenberg, 2004).
Chromatography can be performed using different type of interactions such as
gel filtration, ion exchange, hydrophobic interaction, and affinity chromatography.
Gel filtration chromatography is also known as molecular sieving, gel permeation and
size exclusion chromatography (SEC). The objective of gel filtration is to achieve rapid
separation of molecules based on size. Gel filtration chromatography does not depend
on the adsorption of protein to a solid phase (Wheelwright, 1991).
Hydrophobic interaction chromatography is separations based on the attraction
between hydrophobic groups on the protein and a hydrophobic matrix. The sample is
applied under conditions of high salt concentration and eluted under conditions of low
salt concentration. Hydrophobic interaction chromatography is applicable to most
proteins although the degree of separation is lower than a comparable ion exchange or
affinity chromatography operation (Wheelwright, 1991).
Affinity chromatography relies on a specific interaction between the product
protein and the solid phase to effect separation from contaminants in the feed. Affinity
reactions allow hundred to thousand fold purifications within a single step rather than
relying on relatively small differences between the product and contaminants that lead
to purifications of only few fold improvements. Affinity chromatography is a
concentrating technique which handling large volumes of dilute media and delivering a
small volume of concentrated product (Wheelwright, 1991).
Page 20
9
2.3.2 Membrane Separation
Membrane can be described as an interphase usually heterogenous, acting as a
barrier to the flow of molecular and ionic species present in the liquids and/or vapors
contacting the two surfaces. Separations with membrane do not required additives, and
they can be performed isothermally at low temperatures with less energy consumption
compared to other thermal separation processes. Membrane separation is mainly based
on molecular size but also to a lesser extent on shape and charge.
Microfiltration (MF) and ultrafiltration (UF) are two types of membrane process
that are widely used in large-scale protein purification (Wheelwright, 1991). MF and
UF are differing by the size of the particles they treat. For MF, the range of the particles
is from around 0.05 µm to around 2 µm. For UF, the range of the particles to be treated
is from around 0.2 µm to around 200 nm (Wheelwright, 1991).
MF is commonly used to remove suspended particles from a process fluid and
comprises operations such as the recovery of cells from fermentation broth and the
clarification of lysed-cell slurries. UF on the other hand is an effective technique for
concentrating or separating dissolved molecules of different sizes. In biotechnology,
UF may be used during the initial separation of the cells from the fermentation or
culture medium. It may also be used to separate the cellular fragments from the
medium after the cells are broken (Wheelwright, 1991).
2.3.3 Other Techniques
Besides chromatography and membrane separation method, there are other
techniques that commonly used for protein separation are precipitation, extraction,
centrifugation, electrophoresis and expanded bed adsorption.
In electrophoretic process, the protein molecules are separated in an electric
field at constant pH and current. Two types of electrophoretic methods are isoelectric
focusing and isotachophoresis. In isoelectric focusing, the separation occurs by the pH
Page 21
10
gradient, while in isotachophoresis, the components being separated according to the
conductivities differrence.
Precipitation occurs when the solubility of the protein in solution is reduced
beyond some critical value, or with addition of organic solvents. On the other hand,
extraction is a process in which the protein of interest is transferred from the existing
aqueous phase to another phase, either aqueous or organic.
Centrifugation is a technique used to separate particles from a solution. In the
biological sciences the particles are usually cells, subcellular organelles, large
molecules, or aggregates. There are two types of centrifugation procedures which is
preparative that is used to isolate specific particles, and analytical which involves
measuring physical properties of a sedimenting particle.
2.4 Ion Exchange Chromatography for Protein Fractionation
Ion exchange chromatography is a widely used for protein purification due to its
easy to use and scale-up capabilities. The characteristic of a protein which has net
positive charge in low pH and negative charge in a high pH buffer will be manipulated
in this technique. Isoelectric point (pI) is a point in which the net charge of the protein
is zero. Anion protein is negatively charged and has pH values above pI. Cation
protein is positively charged and has pH values below pI. In ion exchange separations,
the distribution and net charge on the protein’s surface determines the interaction of the
protein with the charged groups on the surface of the chromatography packing material
(Rosenberg, 2004).
The chromatography media, which had covalently attached with positive
functional groups, is referred to an anion exchanger and the one that attached with
negative groups is called as cation exchanger. The charges on the protein and the
packing material must be opposite for the exchange interaction to occur. Proteins that
interact weakly with the ion exchanger will be retained on the column resulting in short
retention times. Meanwhile, proteins that strongly interact with the ion exchanger will
be retained and have longer retention times (Rosenberg, 2004). Figure 2.1 shows a
Page 22
11
guideline to choose cation or anion exchange column based on the operation pH and pI
of the target protein.
Figure 2.1 Guideline for choosing a cation or anion exchange column based on the pI
of the target protein, taken from Rosenberg (2004).
Physical properties such as mechanical strength and flow characteristics,
behavior towards biological substances and capacity is important in selecting the
chromatographic media. Polystyrene, cellulose and polymers of acrylamide and dextran
are basically three major groups of materials used in the construction of ion exchangers.
The advantages of acrylamide and dextran polymers are having molecular sieving
properties, which make it able to be separate on the basis of size and charge (Rosenberg,
2004).
2.5 Mechanism of Ion Exchange Chromatography
Proteins carry both positively and negatively charged groups and are called
“amphoteric”. The charge they carry is dependent upon the pH, with the pH value at
which they have zero net charge termed the isoelectric point, pI. To determine the
optimal conditions for operating an ion exchange separation, the variation in charge of
the protein with pH and the stability of protein across the pH range is important
(Wheelwright, 1991).
At a pH below the pI of protein, the net charge of the protein is positive, and the
protein will bind to a cation exchanger. Meanwhile at a pH above the pI the net charge
Page 23
12
will become a negative, and the protein will bind to an anion exchanger. Table 2.4
describes the conditions under which a protein does or does not adsorb to the ion
exchanger matrix (Wheelwright, 1991).
Table 2.4: Conditions under which a protein binds or does not bind to the ion exchange
matrix (Wheelwright, 1991).
Cation exchanger Anion exchanger Protein net charge
Above pI Does not bind Binds Negative
Below pI Binds Does not bind Positive
Although the net charge of a molecule may be zero at the pI point, the local
charge distribution is not uniform, and areas of positive or negative charge will be found
across the surface of the protein. Therefore, pI alone will not always predict the point at
which the molecule no longer has an affinity for the matrix although it may serve as a
guide for selecting conditions for ion exchange (Wheelwright, 1991).
Proteins are labile molecules and easily denatured by high temperature, extreme
pH, organic solvents, and oxidative atmosphere. It is therefore of paramount
importance that during any chromatographic run these factors be taken into account. If
the protein is an enzyme, simple activity assays can be conducted to determine optimal
conditions for the stability of the protein. It is often a good idea to add reducing agents
such as β-mercaptoethanol or dithiothreitol to prevent oxidation. Speed is also essential
in chromatographic runs. The longer the process the more the protein is exposed to the
oxidative atmosphere. (Rajni and Mattiasson, 2003)
Proteins are made up of amino acids that contain various chemical groups
attached to a peptide backbone. These groups may be positively or negatively charged,
or they may be electrically neutral. The ion exchange resin (solid support matrix)
contains electrically charged species, such as carboxyl or quaternary ammonium groups,
covalently attached. Associated with each linked charge is a counterion of opposite
charge, bound only by ionic attraction (Wheelwright, 1991).
Page 24
13
Charged regions of the protein containing a charge of opposite polarity to the
resin will be attracted to the solid phase. The charged site on the protein will displace
the counterion, and become bound to the resin by ionic interaction. Noncharged species
will not become attached to the resin and may be separated by washing. The protein
bound by ionic interaction can be removed or eluted from the resin by displacement
with an ion having a stronger affinity for the solid phase than the protein does.
(Wheelwright, 1991). Figure 2.2 illustrates the basic idea principle of ion exchange
chromatography.
Figure 2.2 Schematic diagram illustrating mechanism of ion exchange (Wheelwright,
1991). (a) Anion exchange resin with negatively charged counterions. (b) Negatively
charged protein associated with resin. (c) Elution of protein from resin. (d)
Regeneration of resin to original counter ion.
2.5.1 Protein Elution Strategies
Electrostatic forces bind a protein reversibly to an ion exchange column.
Raising the counter-ion (salt) concentration is the most frequently used strategy for
disrupting the electrostatic force between protein and ion exchanger. At low
concentrations, counter-ions (such as Na+ or Cl
-) which are low molecular weight ions,
can be dissociated from an ion exchanger by a protein and at elevated concentrations,
can effectively compete with that protein for binding to the ion exchanger. A salt
elution is simple to perform and is easily reproducible. Two methods exist for
performing a salt elution. In a step elution, the salt concentration is increased in distinct
Page 25
14
steps. A gradient elution utilizes a gradient maker to establish a smooth (continuous)
increase in salt concentration (Bollag et al., 1996).
Besides using a positive gradient of ionic strength, bound proteins can also be
eluted from the column by varying the pH of the eluent. Elution of proteins by the
gradient of pH (continuous or stepwise) is not frequently employed, since some proteins
may not be stable or precipitate at some pHs. Moreover, in conventional ion-exchange
chromatography, a continuous pH gradient is not easy to produce at constant ionic
strength and cannot be achieved by mixing buffers of different pH in linear volume
ratios, since simultaneous changes in ionic strength occur (Ahmed, 2005).
2.6 Advantages of Ion Exchange Chromatography
Most biochemical have unique three-dimensional structures and charge
distributions. Therefore, they can bind with ion-exchange sorbents with distinctly
different affinity. As an example, replacement of two charged amino acid residues in a
protein β-lactoglobulin can result in a different retention time in ion-exchange
chromatography. In addition to its high selectivity, ion-exchange chromatography does
not require potentially denaturing or toxic solvents (Asenjo, 1990).
The salts that are introduced in ion-exchange purification are relatively
harmless. If necessary, they can be removed with a subsequent size-exclusion
chromatography step. Compared to affinity sorbents, ion-exchange sorbents have
higher capacities, can last longer, and are less expensive. Hence, ion-exchange
chromatography has been a popular large-scale (column diameter greater than 2 cm)
separation technique for the purification of amino acids, peptides, enzymes, nucleic
acids, pharmaceuticals, and food products (Asenjo, 1990).
2.7 Preparation of Buffers
A stable pH of the protein environment is important since proteins are extremely
heterogeneous biological macromolecules and their properties can be severely affected
by small changes in hydrogen ion concentration (Ahmed, 2005).